Department of Internal Medicine II, University of Würzburg Medical Center, Oberdürrbacher Str. 6, 97080, Würzburg, Germany.
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.
Anal Bioanal Chem. 2021 Jan;413(2):599-612. doi: 10.1007/s00216-020-03031-7. Epub 2020 Nov 6.
A liquid chromatography tandem mass spectrometry method for the analysis of ten kinase inhibitors (afatinib, axitinib, bosutinib, cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib) in human serum and plasma for the application in daily clinical routine has been developed and validated according to the US Food and Drug Administration and European Medicines Agency validation guidelines for bioanalytical methods. After protein precipitation of plasma samples with acetonitrile, chromatographic separation was performed at ambient temperature using a Waters XBridge® Phenyl 3.5 μm (2.1 × 50 mm) column. The mobile phases consisted of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A and methanol-water (9:1, v/v) with 10 mM ammonium bicarbonate as phase B. Gradient elution was applied at a flow rate of 400 μL/min. Analytes were detected and quantified using multiple reaction monitoring in electrospray ionization positive mode. Stable isotopically labeled compounds of each kinase inhibitor were used as internal standards. The acquisition time was 7.0 min per run. All analytes and internal standards eluted within 3.0 min. The calibration curves were linear over the range of 2-500 ng/mL for afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib, and trametinib, and 6-1500 ng/mL for cabozantinib, dabrafenib, nilotinib, and osimertinib (coefficients of correlation ≥ 0.99). Validation assays for accuracy and precision, matrix effect, recovery, carryover, and stability were appropriate according to regulatory agencies. The rapid and sensitive assay ensures high throughput and was successfully applied to monitor concentrations of kinase inhibitors in patients. Graphical abstract.
一种液相色谱串联质谱法，用于分析人血清和血浆中的十种激酶抑制剂（阿法替尼、阿昔替尼、波舒替尼、卡博替尼、达布拉非尼、仑伐替尼、尼洛替尼、奥希替尼、鲁索替尼和曲美替尼），以便在日常临床常规中应用。该方法按照美国食品和药物管理局和欧洲药品管理局的生物分析方法验证指南进行了开发和验证。在乙腈沉淀血浆样品中的蛋白质后，在环境温度下使用 Waters XBridge® Phenyl 3.5 μm（2.1×50 mm）柱进行色谱分离。流动相由水-甲醇（9:1，v/v）和 10 mM 碳酸氢铵组成的相 A 和甲醇-水（9:1，v/v）和 10 mM 碳酸氢铵组成的相 B 组成。采用 400 μL/min 的流速进行梯度洗脱。采用电喷雾正离子模式下的多重反应监测进行分析物的检测和定量。每种激酶抑制剂的稳定同位素标记化合物被用作内标。采集时间为每次运行 7.0 分钟。所有分析物和内标均在 3.0 分钟内洗脱。阿法替尼、阿昔替尼、波舒替尼、仑伐替尼、鲁索替尼和曲美替尼的校准曲线在 2-500 ng/mL 范围内线性，卡博替尼、达布拉非尼、尼洛替尼和奥希替尼的校准曲线在 6-1500 ng/mL 范围内线性（相关系数≥0.99）。根据监管机构的要求，对准确度和精密度、基质效应、回收率、交叉污染和稳定性的验证试验是合适的。该快速灵敏的测定法可确保高通量，并成功应用于监测患者中激酶抑制剂的浓度。
